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BASILA Report Summary

Project ID: 661715
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - BASILA (Identification and characterization of bacterial effectors that interfere with Transcriptional GeneSilencing in Arabidopsis)

Reporting period: 2015-11-16 to 2017-11-15

Summary of the context and overall objectives of the project

The innate immune response is initiated by the recognition of either evolutionarily conserved pathogen-derived signatures known as Microbe-Associated Molecular Patterns (MAMPs) or the activity of virulence proteins secreted by pathogens called effectors, through PRR receptors or intracellular disease resistance (R) proteins, respectively. Both MAMP-triggered immunity (MTI) and effector-triggered immunity (ETI) rely on a massive transcriptional reprogramming, which is dependent on the accumulation and perception of signalling hormones and results in the differential expression of thousands of immune-responsive genes. Both MTI and ETI were found to be regulated at the post-transcriptional level by RNA silencing, a conserved eukaryotic mechanism that can negatively regulate gene expression in a sequence-specific manner via small RNA guides. RNA silencing also occurs at the transcriptional level through a chromatin-based regulatory mechanism referred to as RNA-dependent DNA methylation (RdDM), which silences repeated sequences and transposable elements to prevent their overexpression and proliferation. Transcriptional gene silencing (TGS) is an ancestral gene regulatory mechanism that operates at the level of chromatin and that is mediated by various histone modifications as well as cytosine DNA methylation. In both plants and animals, DNA methylation transcriptionally silences transposable elements (TEs), repeats, as well as some protein-coding genes that carry TEs/repeats in their vicinity. Notably, the expression of genes that contain transposon sequences in their vicinity can also be modulated by this epigenetic mechanism during their development or during biotic and abiotic stress responses. Numerous factors have been identified as critical components of RdDM, and many remain to be discovered- but this key pathway consists essentially in the production by DICER-Like 3 (DCL3) of 23-24 nt small interfering RNAs that are loaded onto ARGONAUTE 4 (AGO4) to guide DNA methylation. This siRNA-directed DNA methylation is catalyzed by Domain Rearranged Methyltransferase 2 (DRM2) at cytosines in all sequence contexts (CG, CHG and CHH where H is any nucleotide but not a G). During DNA replication, symmetric CG and CHG methylation are then maintained by Methyltransferase 1 (MET1) and the plant specific Chromomethylase 3 (CMT3), while asymmetric CHH methylation is actively perpetuated by RdDM or by CMT2 is a small RNA-independent manner. On the other hand, Arabidopsis encodes DNA glycosylases/lyases that can actively erase DNA methylation, among which Repressor Of Silencing 1 (ROS1), which has been extensively characterized as a negative regulator of RdDM. Whereas DNA methylation has been extensively studied mechanistically and during plant and developmental processes, its dynamics and biological relevance in response to biotic stresses, and particularly during bacterial infections, has just started to be examined and reported. Previous work from the host laboratory has revealed that the disease resistance gene RMG1 and potentially a large number of other targets involved in defence are directly controlled by DNA methylation and active DNA demethylation. This suggested that some bacterial virulence factors may have evolved to interfere with TGS to enable disease. The proposed project aimed at identifying and characterizing such bacterial effectors and their cellular targets. The project was primarily composed of three main tasks:
WP1: Identification of the whole set of Pto DC3000 effectors that interfere with TGS
WP2: Characterization of functionally relevant effector effects on TGS and innate immunity
WP3: Identification and functional characterization of bacterial effector DNA/protein targets in host cells

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

While WP1 allowed the identification of effector candidates that interfere with the transcriptional control of artificial and endogenous RdDM targets. However, it was challenging for us to determine whether the observed effects could be direct or, alternatively, due to the constitutive activation of ETI induced by some effectors or their effects on PTI perception and/or signalling. To rule out this possibility, we have generated additional resources in the course of the BASILA research programme that will be further exploited in the close future by a newly recruited post-doctoral research fellow in the laboratory, who carries on the project. In addition, we have decided to focus our efforts on the characterization of the active DNA demethylase ROS1, which represents a strong candidate target for Pto DC3000 effectors. This significant work provided us with direct ROS1 targets and highlighted a major role for active demethylation in facilitating the transcriptional activation of defense genes, possibly by promoting the accessibility of TFs in their promoter regions. This study, which is currently in preparation for the journal Elife, is a major step towards the identification and characterization of pathogen effectors that directly target the ROS1-dependent demethylation pathway.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We believe that this project will contribute to a deeper understanding of how DNA demethylation controls immune responses in a mechanistic way. This study will likewise contribute to the development of new concepts and resources that will be essential to characterize ‘epigenetic’ effectors that directly interfere with RdDM and/or ROS1 activities and to get insights into their details modes of action. As part of the project, we have also conducted an in-depth characterization of the active demethylase ROS1 in plant immunity and have identified the whole set of immune-responsive genes that are directly controlled by this epigenetic pathway. We are anticipating that these discoveries, which are fundamental in essence, will likely pave the way for future applied research projects that will be conducted in cultivated plants to promote disease resistance against relevant phytopathogen such as Fusarium oxysporum, which is a devastating fungal pathogen that is controlled by active DNA demethylation. Following the BASILA research programme, we have met different European breeding companies and we are currently in discussion with them to initiate translational research projects on this aspect.

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